Fluorescence microscopy is an invaluable tool in biomedical research. Unfortunately, the resolution of conventional fluorescence imaging is limited by diffraction, and as a result the details of biological structures are obscured on lengths smaller than a few hundred nanometers. For example, fundamental aspects of dynamic membrane organization below ~250 nm, remain unclear because of this limitation. In particular, the dynamics of protein recruitment during the early stages of clathrin-mediated endocytosis, an essential process for numerous cellular functions including signaling and growth, cannot be resolved spatially. While certain proteins such as dynamin and endophilin have been linked to the endocytic pathway, the sequence of events occurring during the early stages of endocytosis has yet to be fully understood. Recently, the use of stimulated emission depletion (STED) has provided diffraction-unlimited resolution in fluorescence microscopy. However, simultaneously increasing resolution in all three dimensions has proven less than trivial and phototoxicity to living cells is of concern. The long-term objective of this proposal is to realize a novel super-resolution fluorescence microscope and to apply it to the investigation of the dynamic recruitment of proteins involved in clathrin-mediated endocytosis. The first aim of this project proposes to combine total internal reflection fluorescence (TIRF) microscopy with dual-color STED microscopy to achieve super-resolution imaging in three dimensions at the membrane facing the cover slip. This novel microscope also features less phototoxicity and increases the signal to noise ratio which makes it particularly useful for live cell and dual-color imaging. The second aim of this project proposes to use this setup to investigate protein recruitment during the early stages of clathrin-mediated endocytosis. Using TIRF-STED, protein distributions in living cells will be visualized in real time to test the hypothesis that recruitment of dynamin to clathrin-coated pits is mediated by prior recruitment of endophilin. Specifically, dual-color super-resolution imaging will be used to determine the sequence of events at clathrin-coated pits and compare the evolution of protein cluster morphologies in control and endophilin knockout cells to determine the role of endophilin in the recruitment of dynamin.